A common mechanism by which growth factors regulate cellular proliferation and differentiation is through transmembrane receptors with inducible protein-tyrosine kinase activity (Ullrich and Schlessinger, Cell 61, 203 (1990); Pawson and Bernstein, Trends Gen. 6, 350 (1990)). Indeed the mitogenic effects of growth factors such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) absolutely require the tyrosine kinase activity of their receptors (Chen et al., Nature 328, 820 (1987); Honneger, Mol. Cell. Biol. 7, 4568 (1987); Williams, Science 243, 1564 (1989)). Growth factors induce receptors to cluster, which is followed by intermolecular tyrosine phosphorylation of the oligomerized receptors (Yarden and Schlessinger, Biochemistry 26, 1434 (1987); Boni-Schnetzler and Pilch, Proc. Natl. Acad. Sci. U.S.A. 84, 7832 (1987); Heldin et al., J. Biol. Chem. 264, 8905 (1989)). Autophosphorylation of the PDGF receptor (PDGFR) is important both for its subsequent interactions with substrates and for the induction of DNA synthesis (Kazlauskas and Cooper, Cell 58, 1121 (1989); Coughlin et al., Science 243, 1191 (1989); Kazlauskas et al., Science 247, 1578 (1990)).
A second group of tyrosine kinases, for which Src, Fps, and Abl are the prototypes, are entirely intracellular (Pawson, T., Oncogene 3, 491 (1988)). In the case of the Src-like tyrosine kinase Lck, which is specifically expressed in T cells, the NH.sub.2 -terminal region of the kinase associates with the short cytoplasmic tails of the cell adhesion molecules CD4 and CD8 (Veillette et al., Cell 55, 301 (1988); Rudd et al., Proc. Natl. Acad. sci. U.S.A. 85, 5190 (1988); Shaw et al., Cell 59, 627 (1989)). In addition, Src and the related kinases Fyn and Yes physically associate with, and are phosphorylated by, the .beta.-PDGFR (Kypta et al., Cell 62, 481 (1990)). PDGF stimulation is associated with a three- to five-fold increase in Src kinase activity, which may serve to amplify the tyrosine kinase signal (Kypta et al., Cell 62, 481 (1990); Ralston and Bishop, Proc. Natl. Acad. Sci. U.S.A. 82, 7845 (1985); Gould and Hunter, Mol. Cell. Biol. 8, 3345 (1988)). Hence, the Src-like kinases also appear to participate in signal transduction.
Many structural alterations have been documented for both receptor-like and cytoplasmic tyrosine kinases, which induce constitutive tyrosine kinase activity and simultaneously activate oncogenic potential (Ullrich and Schlessinger, Cell 61, 203 (1990); Pawson and Bernstein, Trends Gen. 6, 350 (1990); Hunter and Cooper, Annu. Rev. Biochem. 54, 897 (1985)). The biological activities of transforming tyrosine kinases, like their normal counterparts, are generally dependent on their kinase activity.
After stimulation with PDGF or EGF several proteins become physically associated with, and phosphorylated by, the activated PDGFR or EGF receptor (EGFR). A number of these receptor-binding proteins have been identified, including phosphoinositide-specific phospholipase C(PLC)-.gamma.1 (Margolis et al, Cell 57 1101 (1989); Meisenhelder et al., ibid., p. 1109), p21.sup.ras GTPase-activating protein (GAP) (Kazlauskas et al., Science 247, 1578 (1990); Kaplan et al., ibid. 61, 121 (1990)), phosphatidylinositol (PI) 3'-kinase (PI3K) (Kazlauskas and Cooper, Cell 58, 1121 (1989); Coughlin et al., Science 243, 1191 (1989)), Src and Src-like tyrosine kinases (Kypta et al., Cell 62, 481 (1990)), and Raf (Morrison et al., ibid. 58, 649 (1989); Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 85, 8855 (1988)). These associated proteins are likely targets of receptor activity.
PLC-.gamma.1 is one of several PLC isoforms that cleaves the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP.sub.2) to the second messengers diacyglycerol and inositol triphosphate, which in turn stimulate protein kinase C and raise intracellular calcium (Rhee et al., Science 244, 546 (1989)). PDGF stimulates PI turnover in cells where PLC-.gamma.1 is the principal PLC isoform (Margolis et al., Cell 57, 1101 (1989); Meisenhelder et al., ibid., p. 1109), and overexpression of PLC-.gamma.1 enhances the accumulation of inositol phosphates in response to PDGF (Margolis et al., ibid. 248, 607 (1990)). Thus, PLC-.gamma. may couple PDGF stimulation to the breakdown of PIP.sub.2.
PI3K phosphorylates the inositol ring of PI in the D-3 position (Whitman et al, Nature 332, 644 (1988)). PI3K activity is associated with a variety of activated tyrosine kinases and correlates with the presence of a tyrosine phosphorylated 85-kilodalton (kD) protein (p85) (Kaplan et al., Cell 50, 1021 (1987); Courtneidge and Heber, ibid., p. 1031; Fukui and Hanafusa, Mol. Cell. Biol. 9, 1651 (1989)). Purified PI3K is a heterodimeric complex that contains p85 and a 110-Kd protein (p110) (Carpenter et al., J. Biol. Chem. 265, 19704 (1990)). The purified p85 subunit has no detectable PI3K activity, but binds tightly to activated PDGFR or EGFR in vitro. PDGF stimulation induces accumulation of PI-3,4-P.sub.2 and PI-3,4,5-P.sub.3, confirming that PI3K is regulated by tyrosine kinases in vivo (Auger et al., ibid. 57, 167 (1989). Stimulation of cells with PDGF or CSF-1 leads to the formation of phosphoinositides which are phosphorylated at the D-3 position of the inositol ring, suggesting that these growth factors stimulate PI 3'-kinase activity in vivo (Auger et al., Cell 57, 167 (1989); Varticovski et al., Nature 352, 699 (1989).
GAP stimulates the ability of p21.sup.ras (Ras) to hydrolyze GTP to GDP (guanosine diphosphate) (B. Margolis et al., ibid, 248, 607 (1990)) and thereby acts as a negative regulator by returning Ras from the active GTP-bound state to the inactive GDP-bound conformation. GAP interacts with the presumed effector region of p21.sup.ras (Adari et al., Science 240, 518 (1988); Cales, Nature (London) 332, 548 (1988)) suggesting that it might also be the Ras target or might modify the association of p21.sup.ras with its target.
Raf is a protein-serine/threonine kinase that complexes with the PDGFR after PDGF stimulation, although it is unclear whether this is a direct interaction (Morrison et al., ibid. 58, 649 (1989); Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 85, 8855 (1988)). Several unidentified polypeptides have also been found to bind to activated PDGFR (Kazlauskas and Cooper, Cell 58, 1121 (1989); Coughlin et al., Science 243, 1191 (1989); Kazlauskas and Cooper, EMBO J. 9, 3279 (1990)).
The proteins that associate with activated growth factor receptors have quite distinct enzymatic properties and are structurally unrelated within their catalytic domains. However, with the exception of Raf they share conserved noncatalytic domains termed Src homology (SH) regions 2, and 3 (see FIG. 1 where 3 represents SH-3 domain; Ras GA the Ras GTPase activating region of GAP; PLC the catalytic sequences of PLC-.gamma.1; gag, retroviral coat protein sequence; CYS, cysteine rich domain of Vav; LEU, leucine-rich region of Vav). cDNAs for two isoforms of p85 of PI3K have been sequenced, and shown to encode closely related proteins with two SH2 domains (Escobede et al., Cell 65, 75, (1991); Otsu et al., Cell 65, 91 (1991); Skolnik et al. Cell 65, 83 (1991)). The SH2 domain is a sequence of .sup..about. 100 amino acids, originally identified in the vFps and vSrc cytoplasmic tyrosine kinases by virtue of its effects on both catalytic activity and substrate phosphorylation (T. Pawson, Oncogene 3, 491 (1988) and I. Sadowski et al., Mol. Cell. Biol. 6, 4396 (1986)).
An SH2 sequence has also been identified in the v-Crk oncoprotein, which complexes with several tyrosine phosphorylated proteins in crk-transformed cells (Mayer et al., Nature 332, 272 (1988); Mayer and Hanafusa, Proc. Natl. Acad. Sci. U.S.A. 87, 2638 (1990)). Most SH2-containing proteins also contain a motif, SH3, which is found independently in several cytoskeletal proteins and may mediate interactions with the cytoskeleton (Pawson, Oncogene 3, 491 (1988); Mayer et al., Nature 332, 272 (1988); Mayer and Hanafusa, Proc. Natl. Acad. Sci. U.S.A. 87, 2638 (1990); Rodaway et al., Nature 342, 624 (1989); Drubin et al., Nature 343, 288 (1990)).
Receptor autophosphorylation may create high affinity binding sites for SH2-containing proteins. Substitution of the Tyr751 autophosphorylation site in the human .beta.PDGF receptor (PDGFR) with phenylalanine decreases PDGF-induced association with PI 3'-kinase (Kazlauskas and Cooper, EMBO J. 9, 327 (1990); Kazlauskas et al., Science 246, 1578 (1990)). A tyrosine phosphorylated peptide spanning the corresponding site of the mouse .beta.PDGFR blocks association of the activated receptor with PI 3'-kinase in vitro (Escobedo et al., Mol. Cell. Biol. 11, 1125 (1991). In general, tyrosine phosphorylation of cellular proteins is important for their stable association with SH2 domains in vitro (Matsuda et al., Science, 248, 1537 (1990); Mayer et al., Proc. Natl. Acad. Sci. 88, 627 (1991)).
The autophosphorylation sites of growth factor receptors are frequently contained in non-catalytic regions located within or adjacent to the kinase domain. The PDGFR subfamily of receptor tyrosine kinases, which includes .alpha. and .beta.PDGFRs, the macrophage colony stimulating factor receptor (CSF-1R) and c-Kit, the receptor for Steel factor, each contain a sequence of between 70 and 100 residues which interrupts the kinase domain. This sequence, called the kinase insert region (KI) is dispensable for kinase activity (Taylor et al., EMBO J. 8, 2029 (1989); Severinsson et al., Mol. Cell. Biol. 10, 801 (1990); Escobedo and Williams, Nature 335, 85 (1988)) but is required for efficient PI 3'-kinase binding (Couglin et al., Science 243, 1191 (1989); Kazlauskas et al., Cell 58, 1121 (1989); Reedijk et al., Mol. Cell. Biol. 10, 5601 (1990); Shurtleff et al., EMBO J. 9, 2415 (1990); Choudhury et al., J. Biol. Chem. 266, 8068 (1991)). The kinase insert contains demonstrated autophosphorylation sites, including Tyr751 in the human .beta.PDGFR (Kazlauskas et al., Cell 58, 1121 (1989)), and Tyr697 and Tyr706 in the mouse CSF-1R (Tapley et al., Mol. Cell. Biol. 10, 2528 (1990); van der Geer and Hunter, Mol. Cell. Biol. 10, 2991 (1990)). Tyr751 and surrounding amino acids in the .beta.PDGFR conform to a proposed consensus binding sequence for PI 3'-kinase, consisting of a phosphorylated tyrosine within the context (Asp/Glu)-(Asp/Glu)-P.Tyr-(Met/Val)-(Pro/Asp/Glu)-Met (Cantley et al., Cell 64, 281 (1991)).
Although they have not been identified as in vivo autophosphorylation sites, the sequence surrounding Tyr721 of the mouse CSF-1R KI and Tyr740 of the human .beta.PDGFR KI also conform to this consensus and are presumptive PI 3'-kinase binding sites. A peptide containing the mouse homologue of human .beta.PDGFR residue 740 inhibits PI 3'-kinase association with the receptor, provided this site is tyrosine phosphorylated (Escobedo et al., Mol. Cell. Biol. 11, 1125 (1991)). Deletion of the CSF-1R KI has been found to result in a profound decrease in the ability of the receptor to associate with PI 3'-kinase in CSF-1 stimulated cells (Reedijk et al., Mol. Cell. Biol. 10, 5601 (1990); Shurtleff et al., EMBO J. 9, 2415 (1990); Coudhury et al., J. Biol. Chem. 266, 8068 (1991)). Preincubation of activated CSF-1R with antibodies raised against a KI peptide containing residues corresponding to 699-719 in mouse CSF-1R has been found to reduce the ability of the receptor to associate with PI 3'-kinase (Downing et al., Mol. Cell. Biol. 11, 2489 (1991)).